A novel method is proposed for quantifying two-dimensional, phase-sensitive, cross-relaxation spectra of proteins. Relative cross-peak volumes are calculated from peak heights and linewidths (measured along the x and y axes). We show that this method gives the same result for isolated peaks as direct volume integration. In the case of moderate peak overlap, our method is less prone to error than volume integration. Computerization of the method is easily implemented and can be used for measuring the massive sets of cross-peak volumes contained in a series of two-dimensional NMR spectra. This approach has enabled us to use a quadratic approximation to the initial build-up rates to determine cross-relaxation rates for 90 proton pairs in a protein, in the laboratory and rotating frames of reference (J. Fejzo, Zs. Zolnai, S. Macura, and J. L. Markley, J. Magn. Reson. 82, 518, 1989). The molecule studied was turkey ovomucoid third domain (OMTKY3), a small (6.1 kDa) globular protein. The cross-relaxation rates in the two frames were analyzed in terms of molecular mobility and interproton distances. The results confirmed that rigid-body isotropic motion with a correlation time of 6 ns holds for OMTKY3. Interproton distances were determined with relative errors of 10-30% and compared with values derived from X-ray crystal structure of this protein.